Involvement of Altered PML/RARalpha and RARbeta Expression in Delayed Restoration of Retinoic Acid Sensitivity in UF-1, an Acute Promyelocytic Leukemia Cell Line Carrying a Patient-Derived Mutant PML/RARalpha(Arg611Trp).

Abstract

[Introduction]Mutations in the PML/retinoic acid receptor alpha (RARa) may be one mechanism of acquired resistance to all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL), but the molecular pathogenesis of this remains unclear. Recently, we reported the delayed restoration of ATRA sensitivity in UF-1, an APL cell line carrying a patient-derived mutant PML/RARa (Leukemia Res, 2004). In this study, we investigated the roles of the mutant PML/RARa and normal RARa in the restored ATRA sensitivity of UF-1.[Methods]After culture in the presence of ATRA or Am80 (RARa-selective RA), the differentiation and apoptosis of UF-1 were evaluated using FACS. RA-dependent transcriptional activity was examined using the luciferase assay. The expression of PML/RARa, RARa, and retinoic acid receptor beta(RARb)was analyzed using RT-PCR and Western-blotting.[Results]UF-1 had little response to ATRA up to day 4, but subsequently showed differentiation and apoptosis in response to ATRA or Am80 at 1 microM, but not 100 nM or lower. In the presence of Am80, UF-1 underwent differentiation similar to that in response to ATRA, while it showed apoptosis at lower degrees than that induced by ATRA. In the luciferase assay, the mutant PML/RARa had little RA-dependent transcriptional activity, but retained a dominant-negative action on normal RARa. Expression of the chimeric protein was decreased in the presence of 1 microM ATRA after day 4, while RARa was preserved. Interestingly, RARb expression was up-regulated in the presence of 1 microM ATRA.[Discussion]The mutant PML/RARa (Arg611Trp), with a reduced ATRA response and a preserved dominant-negative action, caused ATRA resistance of UF-1 during the initial culture period. However, an ATRA-induced decrease in the chimeric protein was thought to be the mechanism by which UF-1 restored ATRA sensitivity after 4 days of culture. Furthermore, the RA signaling that led to apoptosis of UF-1 may be transduced via RARb, which was up-regulated by 1 microM ATRA.